1. Field of the Invention
The present invention relates to branched water-soluble polymers and conjugates formed from these branched polymers.
2. Introduction
The conjugation of the hydrophilic polymers, such as poly(ethylene glycol), abbreviated PEG, also known as poly(ethylene oxide), abbreviated PEO, to molecules and surfaces is of considerable utility in biotechnology and medicine. In its most common form, PEG is a linear polymer terminated at each end with hydroxyl groups:HO—CH2CH2O—(CH2CH2O)n—CH2CH2—OHwhere n typically ranges from about 3 to about 4000. Many end-functionalized derivatives are known in the literature and commercially available. See, for example, Shearwater Polymers, Inc. Catalog “Polyethylene Glycol Derivatives.”
PEG species with a different group at each of the two termini are particularly useful compounds. For example, heterobifunctional PEGs are of use as cross-linking agents. Moreover, PEG molecules that are “capped” at one terminus, e.g., an alkyl group, such as methoxy allow the hydroxyl terminus of the molecule to be converted into any one of a large number of reactive organic functional groups.
Random or block copolymers of ethylene oxide and propylene oxide, shown below, are closely related to PEG in their chemistry, and they can be substituted for PEG in many of its applications.HO—CH2CHRO(CH2CHRO)nCH2CHR—OHin which each R is independently H or CH3.
The formation of conjugates between therapeutically active species and water-soluble polymers has proven a productive strategy for improving the pharmacokinetics and pharmacodynamics of therapeutic agents. See, for example, Dunn and Ottenbrite, “Polymeric Drugs and Drug Delivery Systems:” ACS Symposium Series 469, American Chemical Society, Washington, D.C. 1991. For example, the use of PEG to derivatize peptide therapeutics has been demonstrated to reduce the immunogenicity of the peptides and prolong the clearance time from the circulation. For example, U.S. Pat. No. 4,179,337 (Davis et al.) concerns non-immunogenic peptides, such as enzymes and peptide hormones coupled to polyethylene glycol (PEG) or polypropylene glycol. Between 10 and 100 moles of polymer are used per mole peptide and at least 15% of the physiological activity is maintained.
Many other examples of PEG-peptide conjugates are known in the art. The principal mode of attachment of PEG, and its derivatives, to peptides is a non-specific bonding through a peptide amino acid residue. For example, U.S. Pat. No. 4,088,538 discloses an enzymatically active polymer-enzyme conjugate of an enzyme covalently bound to PEG. Similarly, U.S. Pat. No. 4,496,689 discloses a covalently attached complex of α-1 protease inhibitor with a polymer such as PEG or methoxypoly(ethylene glycol) (“mPEG”). Abuchowski et al. (J. Biol. Chem. 252: 3578 (1977) discloses the covalent attachment of mPEG to an amine group of bovine serum albumin. WO 93/15189 (Veronese et al.) concerns a method to maintain the activity of polyethylene glycol-modified proteolytic enzymes by linking the proteolytic enzyme to a macromolecularized inhibitor. The conjugates are intended for medical applications. U.S. Pat. No. 4,414,147 discloses a method of rendering interferon less hydrophobic by conjugating it to an anhydride of a dicarboxylic acid, such as poly(ethylene succinic anhydride). PCT WO 87/00056 discloses conjugation of PEG and poly(oxyethylated) polyols to such proteins as interferon-β, interleukin-2 and immunotoxins. EP 154,316 discloses and claims chemically modified lymphokines, such as IL-2 containing PEG bonded directly to at least one primary amino group of the lymphokine. U.S. Pat. No. 4,055,635 discloses pharmaceutical compositions of a water-soluble complex of a proteolytic enzyme linked covalently to a polymeric substance such as a polysaccharide.
Another mode of attaching PEG to peptides is through the non-specific oxidation of glycosyl residues on a peptide. The oxidized sugar is utilized as a locus for attaching a PEG moiety to the peptide. For example M'Timkulu (WO 94/05332) discloses the use of a hydrazine- or amino-PEG to add PEG to a glycoprotein. The glycosyl moieties are randomly oxidized to the corresponding aldehydes, which are subsequently coupled to the amino-PEG.
In each of the methods described above, poly(ethyleneglycol) is added in a random, non-specific manner to reactive residues on a peptide backbone. Frequently, derivatization with PEG results in a loss of peptide activity that is directly attributable to the non-selective nature of the chemistries utilized to conjugate the water-soluble polymer.
Another difficulty associated with forming conjugates between water-soluble polymers and biomolecules is the ability of the reactive water-soluble polymer reagent to label the biomolecule at more than one site. Though it is often desirable to include more than one water-soluble polymer moiety per conjugate, the degree of diminution of biomolecule activity is often proportional to the number of polymer moieties bound to the biomolecule. Accordingly, there is interest in obtaining reactive, branched species that include two or more water-soluble polymer moieties per molecule. Through the use of branched molecules, more than one water-soluble polymer can be conjugated to a biomolecule without the necessity of interfering with more than one site on the biomolecule.
Branched polymers based upon poly(ethylene glycol) are known in the art. For example, Greenwald et al. (WO 98/41562) disclose a branched PEG that is based on a 1,3-diamino-2-propanol core. Morpurgo and co-workers discuss the use of branched PEG based on a lysine core is discussed in Appl. Biochem. Biotechnol. 56: 59-72 (1996). A similar lysine-based branched PEG was prepared by Guiotto et al., Bioorg. Med. Chem. Lett 12: 177-180 (2002). Harris et al. (U.S. Pat. No. 5,932,462) also prepared a branched PEG that is based upon lysine. Martinez et al. (U.S. Pat. No. 5,643,575) describe a number of branched PEG species that are based upon various core structures and the conjugation of these species with a biologically active material (U.S. Pat. No. 6,113,906).
Polymers, such as poly(ethylene glycol) are known to exist as heterodisperse populations, which include a range of polymer chain lengths and molecular weights. When preparing therapeutic formulations, it is clearly desirable to utilize polymers with minimal heterodispersity to ensure consistency and reproducibility between preparations. Few methods of preparing mono-disperse PEG samples are known in the art. Loiseau et al. have published a synthesis of well-defined PEG molecules. The method utilizes a protection/deprotection strategy that is less than optimal for the preparation of large quantities of substantially mono-disperse PEGs. Thus, in addition to branched poly(ethylene glycol) polymers, a method for preparing mono-disperse PEG and incorporating the mono-disperse material into the branched polymers would be highly desirable.
The present invention answers the need for both branched water-soluble polymers and mono-disperse PEG species, opening a route to new therapeutic conjugates, e.g., peptide conjugates, and addressing the need for more stable and therapeutically effective therapeutic species. There remains still a need for an industrially practical method for the modification of therapeutic biomolecules with modifying groups such as water-soluble polymers. Of particular interest are methods in which the conjugate has improved properties relative to the unmodified therapeutic agent. The present invention fulfills these and other needs.